Binding assays such as immunoassays, are in widespread use in clinical laboratories for the detection of substances in biological fluids. There is however increasing interest in the development of assays which can be performed without the need for complex analytical techniques and equipment, for example, by a physician in his consulting room or by a patient at home. Such assays are not only more convenient but allow savings in time and expense. Particular applications for which convenient and simple assays and reagent formulations are being sought are the detection of pregnancy and the determination of the fertile period of the menstrual cycle.
Binding assays have been described which employ a strip of material provided with a plurality of reagent zones, in which a developing solution forms a solvent front which passes along the strip by capillary action picking up and facilitating reaction between a sample and assay reagents located at the reagent zones (see for example, British patent specification 1589234). A feature of such strips is the existence of a test location at which, under certain conditions determined by the assay protocol and the sample composition, a labelled reagent becomes immobilised, giving an indication of the assay result. In early assays, the labelled reagent was a binding partner or analogue of the analyte to be measured, labelled with a radioactive isotope. Such assays require instrumentation to detect the level of radioactive label and may present health risk problems. A solution to this has been the use of enzyme labels which produce a characteristic signal (such as a colorimetric signal) with an appropriate substrate.
A significant problem in the design of such so called "dipstick" enzyme-labelled binding assays is the application of the appropriate enzyme substrate in order to produce a detectable signal. The signal may be developed by adding substrate to the appropriate position on the reagent strip after allowing the assay to proceed to completion. Alternatively, the appropriate part of the strip may be removed and chemically analysed. All of these represent steps which would be at least inconvenient, if not impossible for home use of the assay.
In our European Patent Specification No. 225054 we describe a device for conducting competitive and non-competitive enzyme-labelled binding assays which greatly facilitates the use of such assays in the home, with a minimum of manipulative steps. The device may essentially employ an elongate strip, usually of a bibulous paper, and a reservoir. Arranged transversely in order along the strip there may be for example a sample receiving zone, a reagent zone which is impregnated with a suitable enzyme-labelled hapten or antibody for the particular assay protocol in use, and an indicator reagent zone which is capable of immobilising the enzyme-labelled reagent. The reservoir, which may be a rupturable sac, is at the end of the strip adjacent to the sample receiving zone and contains a developing solution which comprises buffer and a signal-producing substrate, together with any necessary cofactors, for the enzyme-labelled reagent. The enzyme and substrate are chosen such that when in contact they produce a signal, such as colour formation which may be readily determined. For other assay protocols the nature of the reagent zone and developing solution may be modified, and additional reagent zones may be present, as appropriate.
By way of example, when the device is used for a competitive hapten assay, a sample is applied to a sample receiving zone and the reservoir is ruptured such that developing solution containing the signal-producing substrate together with any necessary cofactors enters the strip and moves along it by capillary action, picking up the sample and the enzyme-labelled hapten from the respective zones, and transporting them and the signal-producing substrate and any cofactors to the indicator zone where the enzyme labelled hapten and hapten from the sample compete for being sites on an immobilized antibody.
In a particularly useful form of the device the signal-producing substrate, or any necessary cofactors, are chosen such that they migrate more slowly along the strip than the enzyme-labelled reagent. In this way, no signal is formed until the indicator zone when the signal-producing substrate and cofactor meets any bound enzyme-labelled reagent. This differential migration avoids any obscurity in the assay, from incomplete separation of the signal resulting from bound and unbound enzyme-labelled hapten, which can occur when the substrate and enzyme-labelled hapten move along the strip together to the indicator zone. The intensity of the signal at the indicator zone is inversely proportional to the concentration of the hapten in the sample.